Characterization of signal transduction by P2X7 in regulatory T cells by a proteomic approach

ATP released from CD4-helper T cells upon TCR stimulation contributes to the activation of ~1APK signalling through T cells, although the gene encoding P2X7 receptor is comprised in T regs signature genes. Activation of purinergic P2X7 receptor by ATP mediates T regs conversion to pro- infiammatory T cells. thus promoting autoimmuniry.ln this work we identified and characterized the phosphoproteome triggered by BzATP as P2X7 agonist both in stimulated and non-stimulated T regs employing a SlLAC-based proreomic approach in combination with protocols for the enrichment of phosphorylated proteins and high-resolution mass spectrometry. C57BL 6 wild-type tw T) and p2rx7-T regs labelled with different isotopes ( R0K0 or R10K8, respectively) in SILAC media were stimulated with BzATP. After T regs stimulation, cells were lysed and combined prior to enzymatic digestion. Proteolytic digests were fractionated by HILIC and further enriched on a Titansphere Phos-TiO resin. The phosphopeptides were subsequently separated by HPLC coupled online to an LTQ-Orbitrap velos. The mass spectrometer was operated in positive ion mode in data- dependent acquisition mode to alternate between a full scan (m/z 350-2000) in the Orbitrap and subsequent CID MS/MS in the linear ion trap of the 20 most intense peaks from full scan. For identification and quantification of phosphopcptides, Max Quant 1.3.0.5 was used with the mouse UniProt database and Andromeda.